Inhibition of Candida rugosa lipase by saponins, flavonoids and alkaloids

Lipase inhibitors have generated a great interest because they could help in the prevention or the therapy of lipase-related diseases. Therefore, the aim of the work was to evaluate by HPLC, and using Candida rugosa lipase as model, the inhibitory effect of several saponins: beta-aescin, digitonin, glycyrrhizic acid (GA) and Quillaja saponin (QS); flavonoids: 3-hydroxyflavone, 5-hydroxyflavone, (+)-catechin and kaempferol; and alkaloids: aspidospermine, papaverine, physostigmine, pilocarpine, raubasine, rescinnamine, reserpine and trigonelline. The inhibition produced by most of these compounds is described here for the first time. Saponins appeared very active, being beta-aescin and digitonin the most active compounds (IC50 = 0.8-2.4 x 10(-5) M). The inhibitory activity of flavonoids was lower than that of saponins (except GA), and (+)-catechin and kaempferol were the most active. Alkaloids was the most heterogeneous group assayed, varying from rescinnamine, with an IC16 similar to that of digitonin, to papaverine and others which showed almost no inhibition. In conclusion, beta-aescin, digitonin, kaempferol or (+)-catechin, strong lipase inhibitors with a low toxicity and present herbal drugs used for lipase-related diseases such as acne or ulcer, are promising candidates for the prevention or the treatment of these diseases. (c) 2006 Elsevier B.V. All rights reserved

Scalable Lipase-Catalyzed Synthesis of (R)-4-(Acyloxy)pentanoic Acids from Racemic γ-Valerolactone

Conversion of biobased platform chemicals to enantiopure compounds has become topical. We report a straightforward synthesis of 4-(acyloxy)-pentanoic acids from gamma-valerolactone (GVL). An alkaline hydrolysis of GVL is followed by a stereoselective lipase-catalyzed acylation of the sodium salt. Acidic hydrolysis of the acylation product affords (R)-4-(acyloxy)-pentanoic acid and relactonized (S)-GVL. (R)-4-(Propionyloxy)-pentanoic acid and (R)-GVL are obtained with e.r. > 99/1. An additional enzymatic step following a slightly modified process affords (S)-4-(acetyloxy)pentanoic acid with e.r. > 99/1. Simple access to enantiopure 4-(acyloxy)pentanoic acids will stimulate the development of their novel applications, including biobased isotactic polymers

Stereoselective Synthesis of γ‐(Acyloxy)carboxylic Acids and γ‐Lactones Featuring the Switch of Stereopreference of Candida antarctica Lipase B in Sodium γ‐Hydroxycarboxylate Homologues

Scalable protocols of straightforward synthesis of enantiomeric gamma-(acyloxy)carboxylic acids and gamma-lactones are presented. The key step is lipase-catalyzed stereoselective acylation of gamma-hydroxycarboxylic acid sodium salt in organic solvent followed by acidification of the product, extraction and acidic relactonization of the unreacted enantiomer. The mixture of gamma-(acyloxy)carboxylic acid and gamma-lactone is separated either by extraction with solution of sodium bicarbonate or by distillation. A switch of enantioinduction of Candida antarctica lipase B along homologous nucleophiles from R configuration of gamma-hydroxyhexanoic acid salt to S configuration of the C7 and longer-chain homologues has been disclosed. Both enantiomers of gamma-(acyloxy)pentanoic acids; gamma-(acetyloxy)octanoic and -nonanoic acids with S configuration; [(1S,5R)-5-(chloroacetyloxy)cyclopent-2-en-1-yl]acetic acid and enantiomeric gamma-lactones derived from them were prepared with e. r. > 98.5/1.5. The rates of acylation of C5 to C9 homologous salts differ by three orders of magnitude but remain applicable for preparative synthesis by variation of the enzyme loading and reaction time

Catalytic activity of LPL on triglyceride-rich lipoproteins in the presence of fatty acid acceptors and macromolecular crowders.

Measurements were performed using ITC. LPL activity is expressed as heat rate (μJ/s). The substrate mixture contained CM/VLDL (adjusted to 0.5 mM triglycerides), 50 mg/ml BSA (0.75 mM) or 10 mg/ml β-cyclodextrin (8.81 mM) (CYC) and either 10% PEG 6 or 10% dextran 40 (DEX).</p

Interaction of LPL with BSA as studied using ITC, stabilization of LPL, and SPR.

(A) An example ITC thermogram for the titration of BSA into a solution with bLPL, obtained after subtraction of the BSA dilution effect. The concentration of bLPL in the cell was 1.695 μM. (B) Fitted isotherm for the binding between BSA and bLPL from the ITC experiment on panel A. (C) Enzymatic stability of LPL in the presence of BSA as determined with DGGR. 200 nM bLPL or hLPL was incubated for 60 minutes in buffer A at various BSA concentrations. The values are calculated relative to the initial activity of LPL in the same conditions. BSA stabilized both bLPL and hLPL in a concentration-dependent manner. (D) SPR sensograms showing binding of BSA to biotinylated bLPL that was attached to pre-immobilized neutravidin. (E) SPR sensorgrams showing BSA binding to bLPL that was attached to pre-immobilized GPIHBP1. In D and E, BSA concentrations are shown on sensograms. Non-specific binding sensorgrams of BSA to streptavidin and GPIHBP1, respectively, have been subtracted. (F) Plateau values of sensorgrams plotted against BSA concentration. ●—Binding of BSA with 0.1 IU/ml heparin to GPIHBP1-bound bLPL. ▲—Binding of BSA to biotinylated bLPL. ◆—Binding of BSA to 5D2-bound bLPL. Dashed line—1:1 ratio of immobilized bLPL to bound BSA in the experiment with GPIHBP1-bound bLPL. The results indicate that BSA can bind to neutravidin-bound LPL or GPIHBP1-bound LPL but not 5D2-bound LPL.</p

Interaction of albumin with nANGPTL4.

(A) Effect of BSA at various concentrations on nANGPTL4-induced inactivation of LPL during lipolysis of triglyceride-rich lipoproteins. Raw ITC thermograms of lipolysis of CM/VLDL (adjusted to 0.75 mM triglycerides) by LPL in the presence of 100 nM nANGPTL4 and various concentrations of BSA (2 mg/l, 10 mg/ml, 50 mg/ml). The lipolysis rate is expressed as heat rate, μJ/s. The control experiment was performed in the absence of nANGPTL4 and presence of 50 mg/ml BSA. ki represents inhibition rate constant of LPL by nANGPTL4 calculated from the data (n = 2). The results demonstrate that the rate of LPL inactivation by nANGPTL4 is increased at higher BSA concentrations. (B) SPR analysis of binding of various concentrations of HSA to immobilized nANGPTL4. Plateau values of SPR sensorgrams were plotted against HSA concentrations after subtracting non-specific binding. The results indicate that HSA interacts with nANGPTL4.</p

BSA and heparin occasionally form aggregates.

(A) Aggregates of BSA were not observed when 2 mg/ml BSA was applied to a TEM grid. (B) However, aggregates were sometimes observed when 2 mg/ml BSA and 10 IU/ml heparin were mixed together. (C) These BSA/heparin aggregates were also seen in a minority of 200 nM LPL with 2 mg/mL BSA and 10 IU/mL heparin micrographs. Scale bars 100 nm. D—200 nM LPL in 20 mM HEPES, 150 mM NaCl, pH 7.4 buffer. (TIF)</p

Effect of BSA, Triton X-100 or LFHP on LPL activity in the presence of heparin.

(A) 200 nM LPL was incubated for the indicated timepoints with 50 mg/ml BSA, 10 IU/ml heparin or both in buffer A. The remaining activity (expressed as heat rate, μJ/s) was determined with ITC after a single 5 nM LPL injection into human plasma that contained 1.31 mM triglycerides. LPL activity is expressed relative to the initial activity of the experiment that contained both heparin and BSA and the data is presented as mean ± SD of three independent measurements. LPL activity was significantly lower when only BSA or heparin was used. (B) 1 μM LPL was incubated with 10 IU/ml heparin in buffer A and diluted 5-fold to buffer A with heparin and 0.5% Triton X-100, 50 mg/ml BSA or LFHP. The remaining LPL activity was determined in the same manner as panel A. LPL activity was restored equally with every additive.</p